Chitinolytic activity and virulence associated with native and mutant isolates of an entomopathogenic fungus, Nomuraea rileyi

JOURNAL

OF INVERTEBRATE

PATHOLOGY

54, 394-403 (1989)

Chitinolytic Activity and Virulence Associated with Native and Mutant Isolates of an Entomopathogenic Fungus, Nomuraea rileyi’ G.N.EL-SAYED,

T. A. COUDRON,AND C.M.

IGNOFFO

United States Department of Agriculture, Agricultural Research Service, Biological Control of Insects Research Laboratory, P.O. Box 7629, Columbia, Missouri 65205

AND

G. RIBA Institut National de la Recherche Agronomique, Biological Control Research Station, La Miniere, 78280 Guyancourt, France Received July 5, 1988; accepted April 27, 1989 High levels of both endo- and exo-chitinase activity were detected in two virulent isolates but not in one avirulent isolate of Nomuraea rileyi. The greatest difference in chitinolytic activity occurred at germination when endo-chitinase activity in the virulent isolates was 10 to 17 times higher than that in the avirulent isolate, and exo-chitinase activity of the virulent isolates was 15 to 18 times higher than that of the avirulent isolate. Both endo- and exo-chitinase activity were simultaneously expressed in til iSOhteS. 8 1989 Academic Press, Inc. KEY WORDS: Nomuraea rileyi; chitinase; virulence; biological control; entomopathogenic; fungi.

INTRODUCTION

genie fungi (Huber, 1958; Gabriel, 1968; Samsinakova and Misikova, 1973; Ratault and Vey, 1977; Pekrul and Grula, 1979; Rosato et al., 1981; Coudron et al., 1984; Latge et al., 1986; Silva and Messias, 1986; St. Leger et al., 1986a). Of the chitin hydrolases, endo-chitinase and B-Nacetylglucosaminidase (exe-chitinase, also referred to as chitobiase) have been detected in several entomopathogenic fungi, i.e., Beauveria bassiana, Metarhizium anisopliae, Nomuraea rileyi (Coudron et al., 1984, 1985; St. Leger et al., 1986b). Evidence that chitinases are involved in the insect host-pathogen interaction also were reported in other host-parasite systems (Samsinakova et al., 1971; Cornelius et al., 1976; Smith et al., 1981; Charnley, 1984; Brey et al., 1986). However, little is known of the biochemical events responsible for penetration and infection. Our objective was to determine two characteristics, virulence and the level of chitinolytic enzymes during growth of three isolates (one native and two mutants) of the entomopathogenic fungus N. rileyi.

Entomopathogenic fungi are under consideration as potential biological control agents of crop pests. An attractive feature of fungi is that infectivity is by contact and active penetration; most other entomopathogens have to be eaten to be infective. Penetration of the insect integument has been characterized morphologically (Mohamed et al., 1978), ultrastructurally (Boucias and Pendland, 1982; Vey et al., 1982), and histochemically (Gabriel, 1968; Samsinakova et al., 1971; Charnley, 1984; Thorvilson et al., 1985; Brey et al., 1986). Results from these studies suggest a combination of enzymatic degradation and mechanical pressure resulting in penetration and infection of the host. Several possible cuticle-dissolving enzymes have been detected in entomopatho’ This research was supported in part, by U.S. Department of Agriculture Competitive Grant No. 85 CRCR-1-1790 to T.A.C. Mention of a proprietary product in this paper does not constitute a recommendation for use by the U.S. Department of Agriculture or indicate exclusion of other suitable products. 394 0022-2011189 $1.50 Copyright 0 1989 by Academic Press, Inc. AU rights of reproduction in any form reserved.

CHITINASE

MATERIAL Production

AND

AND METHODS

of Fungal Mutants

VIRULENCE

ASSOCIATED

WITH

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deyi

395

YV (= F-83-25, yellow conidia, virulent mutant), and YAV (= F-83-24, yellow conidia, avirulent mutant).

The isolates of N. rileyi used were a naDetermination of Virulence tive green-colored parent isolate (F-83-23), and two yellow-colored auxotrophic muProcedures for producing conidia and tants, one requiring pyridoxal (F-83-24) and blastospores of GV, YV, and YAV, and one requiring methionine (F-83-25). To pro- procedures for topical (treated leaf disc) duce the mutants, conidia of the green vir- and intrahemocoelic experiments were preulent isolate were placed in a flask containviously described (Ignoffo et al., 1982; Iging 10 ml of distilled water and 2 ml of small noffo and Garcia, 1985). In the initial topiglass beads, and separated by agitation cal rate-mortality experiment, first-instar (vertical shaker rotating at 100 strokesimin) larvae of T. ni were exposed to the GV, for 2 min at 20°C. After agitation the conidia YV, and YAV isolates at rates of 0, 3, 30, were exposed to an ultraviolet light (UV) 300, and 3000 conidia/mm2. Each rate was source (wavelength 254 nm, UV-VL 50-W replicated three times using 35 to 50 larvae/ Prolabo, France) in an attempt to produce rate/replicate. The second topical experidifferent colored phenotypic mutants with ment (for all three isolates) was tested at differing levels of virulence. A UV expo300 conidialmm’ using 35 to 50 larvae/ sure rate (13 min) was selected that would replicate and three replicates/isolate. induce 95% of mortality in a conidial susA preliminary intrahemocoelic injection pension containing 10’ conidia/ml (Riba, series (using ca. IO T. ni larvae/dose at 0, 5, 1984). UV-irradiated conidia were plated 50, 500, and 5000 blastospores/third-instar larva) was conducted to determine the most on Sabouraud-maltose agar fortified with 1% yeast extract @MAY) to provide 30 to sensitive dose for comparing all three iso100 colonies per plate. lates. Thereafter, intrahemocoelic injecNone of the auxotrophic mutants were tions were at a dose of 25 blastosporesi complementary when separated by cellolarva. Injection of each isolate was repliphane on a minimal midium. The medium cated three times using 15-25 larvae/ contained the following ingredients: 10 g of replicate. Larvae treated in the same glucose, 1 g of NaNO,, 3 g of sodium manner, but only injected with sterilized glutamate, 0.6 g of MgCl,, 1 g of distilled water, served as a control in these Na2HP04 . 12Hz0 dissolved in 1000 ml of tests. sterile, distilled water. Auxotrophic mutaCulture of Fungi for Determination of tions were identified following Holliday’s Chitinolytic Activity technique (Holliday, 1956). The selection of the native and mutant All three isolates were grown on SMAY isolates for these studies was based on a at 25°C. Conidia harvested from these culpreliminary determination of virulence. In tures were stored at -70°C to preserve vithis determination the parent isolate and ability (Ignoffo et al., 1985). The YAV isoone yellow sibling mutant were both virulate was cultured on SMAY ca. every 4 lent to larvae of Trichoplusia ni (i.e., having weeks to maintain maximum viability and similar LC,, of ca. 40 conidia/mm2). Howavirulence was confirmed via topical treatever, another yellow sibling mutant was ment of T. ni larvae. Insects used in these avirulent to larvae of T. ni (i.e., LC,, studies were reared on a modified semisyngreater than 3000 conidia/mm2). thetic, wheat-germ diet (WIlkinson et al.. For simplicity, the parent and mutant iso- 1972). lates will be subsequently referred to as GV Production of conidia and synchroniza(= F-83-23, green conidia, virulent parent), tion of each isolate was accomplished by

396

EL-SAYED

passing the two virulent isolates (GV and YV) through either T. ni and H. virescens larvae and by passing the avirulent isolate (YAV) on SMAY immediately before the initiation of the biochemical studies. Conidia were suspended, at a concentration of 1.O mg of conidia/ml, in a solution of sterile deionized water containing 0.05% Tween 80 and 1.0 mg/ml of gentamicin (SDWTG). Aliquots (0.1 ml) of the conidial suspension were incubated for 1 hr at 25°C and used to inoculate disposable Petri dishes (100 mm diam.) containing 15 ml of sterilized SMAY. Aliquots of the conidial suspensions were also used to inoculate pads of glass wool impregnated with hemolymph from T. ni. Hemolymph was collected aseptically from early fifth-instar larvae and stored at 4°C in test tubes containing the antioxidant phenylthiocarbamide, at 0.1 mg/ml, and centrifuged at 15OOg for 20 min immediately before use. The supernatant was absorbed onto the glass wool in a 50mm Petri dish. Inoculated plates were incubated at 25°C and subsamples were collected and assayed over a 30-day growth period. Any contaminated plate was discarded. Conformation of synchrony was necessary for the correlation of enzymatic activity with growth stages of the fungi (Coudron et al., 1984; Dillon and Charnley, 1985). Phase microscopy (640x) was used to check uniformity in growth and to establish defined growth stages. Less than 10% deviation from the mean obtained for the enzymatic measurements at different developmental stages confirmed synchrony of the cultures. Germination was defined as the time when 90% of the conidia had a protuberance one-half the diameter of the conidium in length. Elongation of the protuberance greater than one-half the diameter of the conidium in length was defined as the germ tube stage. Blastospore stage (the yeast-cell-like stage where cells are produced by budding from the extended germ tube) generally occurred within 12 hr of the germ tube formation. Subsequent develop-

ET AL.

mental stages were defined as follows: mycelium (filamentous structures within the insect, usually branched), early conidiophore (highly branched mycelium with initial formation of fruiting bodies); and sporulation (formation of conidia exterior on the insect). Enzymatic Activity and Protein Content Preparation of supernatant stock. Two groups of 200 plates were inoculated at separate times for each isolate to ensure that adequate material would be available for sampling. A total of six plates per replicate (three plates taken at 12-hr intervals) over a period of 30 days was used to establish a profile of enzymatic activity. Plates from the two groups were stored at -20°C until all the replicates at each time interval were collected. A brass cork borer (5 mm diam.) was used to cut 10 discs from each plate. The discs were placed in 3 ml of universal buffer, pH 5.1 (Frugoni, 1957), equilibrated at room temperature. Each sample of 10 discs, was sonicated (40 W at 20 kHz, for 30 set); the resulting suspension was homogenized at 5000 t-pm for 2 min in a 50-ml capacity stainless steel chamber (Sorvall, Omni-mixer); and the homogenate was centrifuged at 28,000g for 15 min at 4°C (Sorvall automatic refrigerated centrifuge, rotor SS-34). The supernatant was decanted and saved; the pellet was resuspended in universal buffer and centrifuged at 2OOOgfor 15 min. The second supernatant was combined with the first and the mixture brought to a final volume of 5.0 ml with the addition of universal buffer. The pellets were discarded. This supernatant mixture was the stock solution subsequently used to determine endo- and exo-chitinase activity and protein content. Determination of endo-chitinase activity. Degradation of chitin by the chitinolytic enzyme complex in entomophathgenic fungi is a two-step process (Coudron et al., 1984). The first step, hydrolysis of the long polymer of p-N-acetylglucosamine (NAG), is accomplished by the endo-chitinase (Endo-

CHITINASE

AND

VIRULENCE

ChA) enzyme and is the rate-limiting step in the degradation sequence. Therefore, endochitinase activity can be measured by monitoring the production of N-acetylglucosamine in the presence of excess exochitinase (Exo-ChA). For these studies, Endo-ChA activity was determined by using the p-dimethylaminobenzaldehyde (DMAB) assay (Reissig et al., 1955). Modifications were made to enhance the sensitivity of the assay and to reduce interference from reagents (Coudron et al., 1984). The following procedure was used: A l.Oml aliquot of the supernatant stock (PH 5.1) was incubated (37°C) with 10 mg of pulverized crystalline crustacean chitin (Sigma Chemical Co.) for 4% hr with continuous shaking. The incubation was terminated by boiling for 10 min and the suspended chitin was pelleted by centrifugation at 15OOg for 10 min. A 200-k.1 aliquot of the incubationsolution supematant was diluted with 35 ~1 of borate buffer (0.8 M at pH 9.6), heated to boiling for 10 min, and cooled. Then 0.9 ml of DMAB reagent (0.67 M DMAB in glacial acetic acid: 10 M HCl, lO:O.l, v:v) was added; the sample incubated at 37°C for 20 min, cooled, and the absorbance measured at 585 nm. Enzyme activity was determined by conversion of the absorbance values to NAG concentration using a standard curve. One unit of activity was equivalent to the production of 1 Fmol of reaction product (NAG) per minute at 37°C. ChA is expressed as micromoles of product per milligram of protein to correct for increases in biomass during the development of the fungi (Coudron et al., 1984), the chitinase enzymes secreted by entomopathogenic fungi remain active for several days (unpublished data). The ratio micromoles of product per milligram of protein also provides a correction for activity amplification from residual enzyme produced during an earlier growth stage. Determination of exo-chitinase activity. Exo-ChA was determined spectrophotometrically by measuring the amount of p-

ASSOCIATED

WITH

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397

nitrophenol (pNP) released when the supernatant stock was incubated with the synthetic substrate p-nitrophenol-2acetamido-2-deoxy-P-D-glucopyranoside (pNADG) (Sigma Chemical Co). Optimal conditions for enzyme activity were selected as previously described (Coudron et al., 1984). The following procedure was used: Five hundred microliters of supematant stock was diluted with 700 p,l of 0.28 mglml of pNADG in universal buffer @H 5.1) and incubated for 1 hr at 37°C. The reaction was terminated by adding 1.2 ml of 0.02 M NaOH and the absorbance measured at 400 nm. Readings were converted to pNP concentration using a standard curve and the use of the value EM = 1.8 x lo4 (Bender et al., 1967). One unit of enzyme activity was defined as that amount capable of liberating 1 pmol of reaction product (p-nitrophenol) from the substrate per minute at 37°C. Determination of protein. Protein was measured using bovine serum albumin as a standard and a protein assay (Bio-Rad), based on a Coomassie brilliant blue dye binding procedure of Bradford (1976). Absorbance was measured at 595 nm. Protein measurements in fungi cultures were corrected for background values obtained from blank SMAY plates. RESULTS Determination

of virulence

All larvae of T. ni topically exposed to either GV or YV at 3000 conidia/mm2 died of a Nomuraea-induced mycosis (Table 1). In contrast, no larvae exposed to isolate YAV died of a mycosis. Nomuraeainduced mycoses by the parent GV and mutant YV were obtained even at the lowest rate of 3 conidia/mm2. In addition, there was no apparent difference in mortality of T. ni larvae between the parent GV and the mutant YV. The LC,, (graph-interpolated) for larvae of T. ni for the parent GV and the mutant YV and YAV isolates were 45. 38,

398

EL-SAYED

ET AL.

TABLE SUSCEPTIBILITY

1

OF LARVAE OF Trichoplusia ni TO TOPICAL APPLICATION OF CONIDIA ISOLATES AND ONE NATIVE ISOLATE OF Nomuraea rileyi”

Mutant virulent (YV)

Native virulent (GV)b Dose conidia per mm*

Number used

3OOcl 300 30 3 0

142 137 145 149 150

% Mortality x

(SEM)

x

Two

MUTANT

Mutant avirulent (YAV)

% Mortality

Number used

FROM

(SEM)

100.0

-

137

100.0

-

89.3 42.7 2.7 0.0

(2.3) (6.1) (0.7) -

140 142 144 149

90.7 50.0 6.3 0.0

(4.4) (3.8) (1.2) -

% Mortality

Number used

x

147 147 132 140

0.0 0.0 0.0 0.0

144

0.0

a Average of three replicates of 35 to 50 larvae/dose/replicate. b GV, green virulent isolate; YV, yellow virulent isolate; YAV, yellow avirulent isolate.

injected addition, obtained on T. ni

and greater than 3000 conidia/mm2, respectively. When all three isolates were topically exposed at a single rate (300 conidial mm2) the parent GV and mutant YV induced 98% mortality of T. ni larvae. No mycosis was detected in larvae exposed to the YAV isolate. Results similar to the topical treatments were obtained when T. ni larvae were injected with blastospores (Table 2). Injections of 25 blastospores/larva of GV, YV, or YAV induced 87,98, and 0% larval mortality, respectively. Although no mortality with YAV occurred at 25 blastosporesl larva, mortality did occur when larvae were

Determination of Chitinolytic Activity (ChA) A negligible quantity of soluble protein was detected in GV and YV during the first 2 days; however, the protein content in YAV increased three-fold during the first day, followed by a decrease prior to the blastospore stage (Fig. 1). The production of soluble protein increased rapidly in all three isolates at the onset of the blastospore

TABLE SUSCEPTIBILITY OF LARVAE CONIDIA OR BLASTOSWRES,

OF Trichoplusia RESPECTIVELY,

with 5000 blastospores/larva. In growth to the mycelial stage was when blastospores were cultured hemolymph.

2

ni TO TOPICALLY AND INTRAHEMOCOELICLY ADMINISTERED OF Two MUTANT ISOLATES AND ONE NATIVE ISOLATE OF Nomuraea rileyi

Topical”

Isolate

Number used

Native virulent (GV) Mutant virulent (YV) Mutant avirulent (YAV)

128 141 136

Injectionb % MortalityC x (SEM) 98.4 (0.7) 98.6 (0.7)

0.0

Number used 54 59 65

% Mortalityd

x (SEM) 87.0 (9.9) 98.3 (0.6) 0.0

a Rate of 300 conidia/mm’. b Dose of 25 blastospores!Iarva. c Average of three replicates of 35 to 50 larvae/replicate. No mortality in controls (145 larvae). d Average of three replicates of 15 to 25 larvae/replicate. No mortality in injected controls (61 larvae) or noninjected control (74 larvae). e GV, green virulent isolate; YV, yellow virulent isolate; YAV, yellow avirulent isolate.

CHITINASE

AND

VIRULENCE

ASSOCIATED

WITH

N.

399

rileyi

TABLE

3

AVERAGE NUMBER OF DAYS TO ATTAIN VARIOUS DEVELOPMENTAL STAGES FOR ONE NATIVE ISOLATE AND Two MUTANT ISOLATES OF N. rileyi

Developmental stages”

FIG. 1. Soluble protein content of supematant stock for Nomuraea rileyi isolates, GV, YV, and YAV. GV, green virulent parent (-); YV, yellow virulent mutant (-----); YAV, yellow avirulent mutant (- - -). Time of occurrence of each developmental stage is given in Table 3.

stage through the onset of the mycelial stage. Thereafter, soluble protein increased slowly for YV and YAV and decreased slowly for GV. The soluble protein content increased in GV and YAV but decreased slightly for YV during the early conidiophore stage. After sporulation, protein content increased significantly for YAV, but only gradually increased for GV and YV. Refer to Table 3 for the designation of time periods required to attain each of these growth stages. Changes in the Endo-ChA and Exo-ChA appeared to be age corelated. The timing of Endo-ChA markedly varied for the three isolates (Fig. 2, Table 4). Endo-ChA increased six- and ninefold during germination for YV and GV, respectively. By comparison, total Endo-ChA in the YAV only doubled during germination. All isolates increased in Endo-ChA after germination and prior to the formation of blastospores. The Endo-ChA for YV declined rapidly during the late blastospore and early mycelium stages for YV but increased slightly for GV and YAV. A gradual decline in Endo-ChA occurred in all three isolates during the early conidiophore and sporulation stages.

Days post-inoculation GVb

YV

YAV

Conidia‘ 0 0 0 Germination/ germ tube 2.0 2.0 1.5 Blastospore 3.0 3.0 2.5 Mycelium 5.0 5.0 4.5 Early-conidiophore 9.0 8.5 8.5 Sporulation 15.0 15.0 13.0 ~__ u The developmental stages are defined and described in the text. b GV, green virulent isolate; YV, yellow virulent isolate; YAV, yellow avirulent isolate. ’ Inoculation time.

The pattern of Exo-ChA was similar to the Endo-ChA pattern (Fig. 3, Table 4). A pronounced increase of Exo-ChA was measured prior to germination and continued into the blastospore stage. The activity continued to decline during the mycelium, early conidiophore, and sporulation stages for the GV and YV. Isolate YAV activity was negligible prior to germination, slowly 100

,

90 80 i

clays

2. Endo-chitinase activity expressed as p,mol of NAGimg of soluble protein during the developmental stages for Nomuraea rileyi isolates, GV, YV, and YAV. GV, green virulent parent (---); YV, yellow virulent mutant (-----); YAV. yellow avirulent mutant (- - ---I. Time of occurrence of each developmental stage is given in Table 3. FIG.

400

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ET AL.

TABLE

4

&ado-CHITINASE AND EXO-CHITINASE ACTIVITY AT VARIOUS DEVELOPMENTAL STAGES FOR THREE DIFFERENT

ISOLATES

Total enzyme activity @mole productimg protein)b

Developmental stage@

GVd

YV

tube

2.26 (1.00) 18.81 (2.77) 27.86 (1.42) 43.98 (1.59)

31.09 (1.36) 14.50 (0.70)

5.65 34.71 61.09 35.22 27.65 19.34

Specific activity ratio’ YAV

Endo-chitinase Conidia’ Germination/germ Blastospore Mycelium Early-conidiophore Sporulation

OF N. rileyi

[GV]/[YAV]

[YV]/[YAV]

4.87 16.93 4.97 3.95 2.13

[NAG]

(2.07) (2.07) (2.19) (1.88)

1.16 (0.73) 2.05 (0.73) 12.30 (1.10) 8.92 (1.08)

(1.18)

12.96 (0.50)

1.95 9.18 2.27 4.93 2.40

(1.13)

7.40 (0.41)

1.96

2.61

Exo-chitinase [pNP] Conidia’ Germination/germ tube Blastospore Mycelium Early-conidiophore Sporulation

0.74 (0.02)

0.25 (0.09)

10.41 (0.31)

8.81 (0.72)

23.67 (0.69) 33.95 (0.67) 29.28 (0.72)

12.58 (0.59)

58.98 30.90 32.08 24.05

(1.66) (0.28) (0.61) (0.31)

0.33 (0.33) 0.56 (0.26) 3.84 (0.86) 4.42 (1.11)

11.95 (1.07) 7.91 (0.65)

2.24 18.59 6.16 7.68 2.45 1.59

0.76 15.73 15.36 6.99 2.69 3.04

o Recorded every 12 hr for 30 days. b Each point represents the mean (SEM) value of enzyme activity for each developmental stage; determined from two experiments. ’ Ratios were calculated using total enzyme activity. d GV, green virulent isolate; YV, yellow virulent isolate; YAV, yellow avirulent isolate. e Conidia at inoculation,

increased during the blastospore and early conidiophore stages, and then gradually declined during sporulation. Exo-ChA in YAV was considerably lower than that of the virulent isolates. Total Endo-ChA for YAV was approximately 25 to 30% of that for the two isolates tested (Table 4). The ratio of Endo-ChA at the time of germination for GV vs YAV and YV vs YAV reached its highest level of 9 and 17, respectively. The highest ratio of Exo-ChA for GV vs YAV and YV vs YAV also occurred at germination and was of 19 and 16, respectively (Table 4). DISCUSSION

Ours is the first quantitative study comparing virulence and ChA in isolates of the entomopathogenic fungus N. rileyi. Silva and Messias (1986) reported qualitative comparisons of the proteolytic, lipolytic, and amylolytic activity of virulent mutants

and revertants isolated from the fungus M. anisopliae. Their assay technique, however, did not correlate expression of enzymatic activity and virulence to the rate of fungal development. Expression of enzymatic activity depends on the stage and rate of fungal development (Coudron et al., 1984, 1985; St. Leger et al., 1986~). An accurate comparison of the ChA should reflect an accounting for the growth stage as well as the time after inoculation (Ricciardi et al., 1974; Coudron et al., 1984; St. Leger et al., 1986c, 1987). The culture conditions used in these experiments were selected to yield a high degree of synchrony in germination and growth. The use of SMAY had several advantages. It was free of contamination from insect components and expressed the full potential of chitinase syntheses; i.e., no substrate induction was observed (Santos et al., 1979; Coudron et al., 1984). It also enhanced the ability of the fungus to over-

CHITINASE

4

<,

2

4

6

8

10

I2

14

AND

16

18

20

22

VIRULENCE

24

26

28

31

FIG. 3. Exe-chitinase activity expressed as pmol of p-nitrophenol (pNP)/mg of soluble protein during developmental stages for Nomuraea riieyi isolates GV, YV, and YAV. GV, green virulent parent (-); YV, yellow virulent mutant (-----); YAV, yellow avirulent mutant (- - -). Time of occurrence of each developmental stage is given in Table 3.

come the potential of catabolite repression of enzymatic activity (Cooper, 1977; Coudron et al., 1984). Synchronization of growth may also provide synchronized penetration for maximum virulence (Hassan and Charnley, 1983; Dilon and Charnley, 1985), which is important when analyzing isolates with varying degrees of virulence. Chitinase enzymes were produced in significant amounts by the virulent isolates GV and YV but not by the avirulent isolate YAV. Two forms of chitinase enzymes (Endo-ChA and Exo-ChA) were produced by all isolates during growth from conidial germination to sporulation. Chitinase activity recorded for GV and YV, during germination, increased at a faster rate than the increase in protein production. As a result, the ChA/protein ratio during germination is as much as 35 times that found in conidia. However, that ratio only doubled in YAV during germination. Rapid amplification in the rate of increase in ChA only occurred in the virulent isolates at the onset of the blastospore stage. High levels of ChA, therefore, were present at a stage critical to penetration of the insect’s integument.

ASSOCIATED

WITH

N.

rileyi

40 I

Results from previous studies (Coudron et al., 1984) showed that chitinolytic levels in three entomopathogenic fungi increased rapidly after germination and continued to increase, reaching a maximum level at or immediately before sporulation. That indicates that chitinolytic activity in these fungi is important to growth as well as potentially needed for penetration. The results presented in this study show that virulent and avirulent isolates of N. rileyi contain chitinolytic activity throughout their growth. However, virulent isolates consistently contain significantly higher levels of activity at the time of penetration which supports their ability to penetrate the chitin-ladened cuticle of the host insect. Although the function and regulation of chitinases is unclear, there is no doubt that these enzymes alter the overall integrity of chitin. The simultaneous expression and similar patterns of both endo- and exochitinases suggest that these enzymes act synergistically and are under common regulatory control. Additional information on the characterization and regulation of chitinases and other enzymes (i.e., proteases, lipases, and chymoelastases) is required before we understand their role in the penetration and infection process of entomopathogenic fungi. ACKNOWLEDGMENTS We express our appreciation to Darcy Shackelford, Clemente Garcia, and James Smith for their technical assistance. Recognition also is given to Dr. M. Elaroussi for his help in the computer analyses. We appreciate the diligence of the following reviewers: Drion G. Boucias, Richard M. Cooper, Karl J. Kramer. Timothy D. Leathers, Jose C. Silva. and Raymond J. St. Leger.

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